The Application of Proteomic Techniques to Fungal Protein Identification and Quantification

Abstract

The number of sequenced genomes has increased rapidly in the last few years, supporting a revolution in bioinformatics that has been leveraged by scientists seeking to analyze the proteomes of numerous biological systems. The primary technique employed for the identification of peptides and proteins from biological sources is mass spectrometry (MS). This analytical process is usually in the form of whole-protein analysis (termed "top-down" proteomics) or analysis of enzymatically produced peptides (known as the "bottom-up" approach). This article will focus primarily on the more common bottom-up proteomics to include topics such as sample preparation, separation strategies, MS instrumentation, data analysis, and techniques for protein quantification. Strategies for preparation of samples for proteomic analysis, as well as tools for protein and peptide separation will be discussed. A general description of common MS instruments along with tandem mass spectrometry (MS/MS) will be given. Different methodologies of sample ionization including matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) will be discussed. Data analysis methods including database search algorithms and tools for protein sequence analysis will be introduced. We will also discuss experimental strategies for MS protein quantification using stable isotope labeling techniques and fluorescent labeling. We will introduce several fungal proteomic studies to illustrate the use of these methods. This article will allow investigators to gain a working knowledge of proteomics along with some strengths and weaknesses associated with the techniques presented.

Identification of in planta-expressed arbuscular mycorrhizal fungal proteins upon comparison of the root proteomes of Medicago truncatula colonised with two Glomus species

“…By directly targeting proteins that are key effectors of biotrophic interactions, proteome analysis may thus unravel some actors of the symbiotic molecular dialog, including those of fungal origin. Although initial proteomic studies conducted on mycorrhizal roots of M. truncatula have experimented difficulties in revealing the intra-radicular accumulation of fungal gene products both at the early or late stages of AM symbiosis (Bestel-Corre et al, 2002, 2004Amiour et al, 2006), many recent advances in bioinformatics and mass spectrometry (MS) have improved the speed and breadth of samples that can be efficiently analysed in large-scale protein profiling experiments in plants and fungi (Oeljeklaus et al, 2008;Rohrbough et al, 2007). With regard to AM symbiosis, systematic nanoscale capillary liquid chromatography-MS/MS (LC-MS/MS) has enlarged the coverage of mycorrhiza-related proteins along with the identification of AM fungal proteins (Aloui et al, 2009;Recorbet et al, 2009).…”

Optimization of a protein extraction technique for fungal proteomics

“…This leads to a diffi culty in detection of low-abundance proteins in the presence of highly abundant ones. Unlike RNA-based methods of transcript amplifi cation, sample protein levels cannot be increased to facilitate analysis of low-abundance proteins [7]. Membrane proteins play a crucial role in many key processes, including exchange and recognition processes.…”